Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens

To detect agrobacteria causing crown gall disease of grapevine novel virulence and oncogene specific primer combinations were tested on Agrobacterium vitis and Agrobacterium tumefaciens strains including most opine types found in grapevines. Reproducible detection of all the tested pathogens in a single reaction was only possible with multiplex PCR using mixtures of virulence-, or oncogene specific primers. A primer combination including pehA, virF and virD2 gene-specific oligonucleotides amplified the corresponding fragments from nearly all strains included and distinguished A. vitis and A. tumefaciens strains carrying octopine or nopaline pTis and A. vitis vitopine strains. A second set of primers designed to amplify the T-DNA auxin genes iaaH and iaaM detected all of the tested pathogens and, as in the case of virF-, and virD2-specific primers, A. vitis vitopine strains formed also a distinct group. These data were further confirmed using opine synthase-, or 6b gene-specific primers that also allowed the identification and distinction of octopine and nopaline as well as vitopine isolates of A. vitis. Thus, a wide range of agrobacteria occurring on grapevine were detected and identified. On the other hand, our results confirm that vitopine-type agrobacteria form a distinct group within the genus Agrobacterium.

Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens

Summary To detect agrobacteria causing crown gall disease of grapevine novel virulence and oncogene specific primer combinations were tested on Agrobacterium vitis and Agrobacterium tumefaciens strains including most opine types found in grapevines. Reproducible detection of all the tested pathogens in a single reaction was only possible with multiplex PCR using mixtures of virulence-, or oncogene specific primers. A primer combination including pehA, virF and virD2 gene-specific oligonucleotides amplified the corresponding fragments from nearly all strains included and distinguished A. vitis and A. tumefaciens strains carrying octopine or nopaline pTis and A. vitis vitopine strains. A second set of primers designed to amplify the T-DNA auxin genes iaaH and iaaM detected all of the tested pathogens and, as in the case of virF-, and virD2-specific primers, A. vitis vitopine strains formed also a distinct group. These data were further confirmed using opine synthase-, or 6b gene-specific primers that also allowed the identification and distinction of octopine and nopaline as well as vitopine isolates of A. vitis. Thus, a wide range of agrobacteria occurring on grapevine were detected and identified. On the other hand, our results confirm that vitopine-type agrobacteria form a distinct group within the genus Agrobacterium. K e y w o r d s

Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance

Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. 'Richter 110' rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twentyone transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58

Aedes koreicus, a vector on the rise: Pan-European genetic patterns, mitochondrial and draft genome sequencing

25openYesBackground The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species’ biology, behavior, and dispersal patterns were poorly investigated to date. Methodology/Principal findings To understand the species’ population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. Conclusions/Significance Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of ‘Single Nucleotide Polymorphism’ markers. Considering its public health importance, it is crucial to further investigate the species’ population genetic dynamic, including a larger sampling and additional genomic markers.Kurucz, Kornélia; Zeghbib, Safia; Arnoldi, Daniele; Marini, Giovanni; Manica, Mattia; Michelutti, Alice; Montarsi, Fabrizio; Deblauwe, Isra; Van Bortel, Wim; Smitz, Nathalie; Pfitzner, Wolf Peter; Czajka, Christina; Jöst, Artur; Kalan, Katja; Šušnjar, Jana; Ivović, Vladimir; Kuczmog, Anett; Lanszki, Zsófia; Tóth, Gábor Endre; Somogyi, Balázs A; Herczeg, Róbert; Urbán, Péter; Bueno-Marí, Rubén; Soltész, Zoltán; Kemenesi, GáborKurucz, K.; Zeghbib, S.; Arnoldi, D.; Marini, G.; Manica, M.; Michelutti, A.; Montarsi, F.; Deblauwe, I.; Van Bortel, W.; Smitz, N.; Pfitzner, W.P.; Czajka, C.; Jöst, A.; Kalan, K.; Šušnjar, J.; Ivović, V.; Kuczmog, A.; Lanszki, Z.; Tóth, G.E.; Somogyi, B.A.; Herczeg, R.; Urbán, P.; Bueno-Marí, R.; Soltész, Z.; Kemenesi, G

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of SARS-CoV-2 genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three available genomic nomenclature systems for SARS-CoV-2 to all sequence data from the WHO European Region available during the COVID-19 pandemic until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation. We provide a comparison of the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2.Peer reviewe

Novel pathogen-specific primers for the detection of Agrobacterium vitis and Agrobacterium tumefaciens

Summary To detect agrobacteria causing crown gall disease of grapevine novel virulence and oncogene specific primer combinations were tested on Agrobacterium vitis and Agrobacterium tumefaciens strains including most opine types found in grapevines. Reproducible detection of all the tested pathogens in a single reaction was only possible with multiplex PCR using mixtures of virulence-, or oncogene specific primers. A primer combination including pehA, virF and virD2 gene-specific oligonucleotides amplified the corresponding fragments from nearly all strains included and distinguished A. vitis and A. tumefaciens strains carrying octopine or nopaline pTis and A. vitis vitopine strains. A second set of primers designed to amplify the T-DNA auxin genes iaaH and iaaM detected all of the tested pathogens and, as in the case of virF-, and virD2-specific primers, A. vitis vitopine strains formed also a distinct group. These data were further confirmed using opine synthase-, or 6b gene-specific primers that also allowed the identification and distinction of octopine and nopaline as well as vitopine isolates of A. vitis. Thus, a wide range of agrobacteria occurring on grapevine were detected and identified. On the other hand, our results confirm that vitopine-type agrobacteria form a distinct group within the genus Agrobacterium. K e y w o r d s

Methylene Blue Is a Nonspecific Protein–Protein Interaction Inhibitor with Potential for Repurposing as an Antiviral for COVID-19

We have previously identified methylene blue, a tricyclic phenothiazine dye approved for clinical use for the treatment of methemoglobinemia and for other medical applications as a small-molecule inhibitor of the protein–protein interaction (PPI) between the spike protein of the SARS-CoV-2 coronavirus and ACE2, the first critical step of the attachment and entry of this coronavirus responsible for the COVID-19 pandemic. Here, we show that methylene blue concentration dependently inhibits this PPI for the spike protein of the original strain as well as for those of variants of concern such as the D614G mutant and delta (B.1.617.2) with IC50 in the low micromolar range (1–5 μM). Methylene blue also showed promiscuous activity and inhibited several other PPIs of viral proteins (e.g., HCoV-NL63–ACE2, hepatitis C virus E–CD81) as well as others (e.g., IL-2–IL-2Rα) with similar potency. This nonspecificity notwithstanding, methylene blue inhibited the entry of pseudoviruses bearing the spike protein of SARS-CoV-2 in hACE2-expressing host cells, both for the original strain and the delta variant. It also blocked SARS-CoV-2 (B.1.5) virus replication in Vero E6 cells with an IC50 in the low micromolar range (1.7 μM) when assayed using quantitative PCR of the viral RNA. Thus, while it seems to be a promiscuous PPI inhibitor with low micromolar activity and has a relatively narrow therapeutic index, methylene blue inhibits entry and replication of SARS-CoV-2, including several of its mutant variants, and has potential as a possible inexpensive, broad-spectrum, orally bioactive small-molecule antiviral for the prevention and treatment of COVID-19

Genetic and Genomic Approaches for Adaptation of Grapevine to Climate Change

The necessity to adapt to climate change is even stronger for grapevine than for other crops, because grape berry composition—a key determinant of fruit and wine quality, typicity and market value— highly depends on “terroir” (complete natural environment), on vintage (annual climate variability), and on their interactions. In the same time, there is a strong demand to reduce the use of pesticides. Thus, the equation that breeders and grape growers must solve has three entries that cannot be dissociated: adaptation to climate change, reduction of pesticides, and maintenance of wine typicity. Although vineyard management may cope to some extent to the short–medium-term effects of climate change, genetic improvement is necessary to provide long-term sustainable solutions to these problems. Most vineyards over the world are planted using vines that harbor two grafted plants’ genomes. Although this makes the range of interactions (scion-atmosphere, rootstock-soil, scion-rootstock) more complex, it also opens up wider possibilities for the genetic improvement of either or both the grafted genotypes. Positive aspects related to grapevine breeding are as follows: (a) a wide genetic diversity of rootstocks and scions that has not been thoroughly explored yet; (b) progress in sequencing technologies that allows high-throughput sequencing of entire genomes, faster mapping of targeted traits and easier determination of genetic relationships; (c) progress in new breeding technologies that potentially permit precise modifications on resident genes; (d) automation of phenotyping that allows faster and more complete monitoring of many traits on relatively large plant populations; (e) functional characterization of an increasing number of genes involved in the control of development, berry metabolism, disease resistance, and adaptation to environment. Difficulties involve: (a) the perennial nature and the large size of the plant that makes field testing long and demanding in manpower; (b) the low efficiency of transformation, regeneration and small size of breeding populations; (c) the complexity of the adaptive traits and the need to define more clearly future ideotypes; (d) the lack of shared and integrative platforms allowing a complete appraisal of the genotype-phenotype-environmental links; (e) legal, market and consumer acceptance of new genotypes. The present chapter provides an overview of suitable strategies and challenges linked to the adaptation of viticulture to a changing environment